Various scientific and patent publications are referred to herein. Each is incorporated by reference in its entirety.
Biomolecules such as DNA or RNA are long molecules composed of nucleotides, whose linear sequencing is directly related to the genomic and post-genomic expression information of the organism.
Biomolecules such as DNA or RNA are long molecules composed of nucleotides, whose linear sequencing is directly related to the genomic and post-genomic expression information of the organism.
In many cases, the mutation or rearrangement of the nucleotide sequences during an individual's life span leads to disease states such as genetic abnormalities or cell malignancy. In other cases, the small amount of sequence differences among each individual reflects the diversity of the genetic makeup of the population. Because of this, different people have different disease predisposition or respond differently to environmental stimuli and signals such as stress or drug treatments. As an example, some patients experience a positive response to certain compounds while others experience no effects or even adverse side effects. Another area of interest is the response of biomolecules such as DNA to environmental toxins or other toxic stimuli such as radiation. Toxic stimuli can lead to programmed cell death (apoptosis), a process that removes toxic or non-functioning cells. Apoptosis is characterized by morphological changes of cells and nuclei and is often accompanied by the degradation of chromosomal DNA.
Areas of population genomics, comparative/evolution genomics, medical genomics, environmental or toxicogenomics, and pharmacogenomics studying genetic diversity and medical pharmacological implications require extensive sequencing coverage and large sample numbers. Knowledge generated from such study would thus be especially valuable to the health care and pharmaceutical industry. Cancer genomics and diagnostics in particular study genomic instability events leading to tumorigenesis. All these fields would thus benefit from technologies enabling fast determination of the linear sequence, structural pattern changes of elements/regions of interests on biopolymer molecules such as nucleic acids, or epigenetic biomarkers such as methylation patterns along the biopolymers.
Most genome or epigenome analysis technologies remain too tedious or expensive for general analysis of large genomic regions or for a large population. Thus, to achieve the goal of reducing the genomic analysis cost by at least four orders of magnitude, the so-called “$1000 genome” milestone, new paradigm shift technologies enabling direct molecular analysis methods are desirable. See “The Quest for the $1,000 Human Genome”, by Nicholas Wade, The New York Times, Jul. 18, 2006.
Additionally, it takes on average 7-10 years and 800 million dollars to bring a new drug to market. Accordingly, pharmaceutical companies are seeking a safer and economical ways to hasten drug development while minimizing the toxicity failure rate.
Drug compound toxicity can result in damages to DNA in the form of gene mutations, large scale chromosomal alterations, recombination and numerical chromosomal changes. Genotoxicity tests are in vitro and in vivo tests done in bacterial, mammalian cells and animals to detect compounds that induce such damages directly or indirectly by various mechanisms. The positive compounds could be human carcinogens and/or mutagens that induce cancer and/or heritable defects. A drug can be toxic at different levels, but drug-induced mutagenesis of DNA, such as germ line mutations, underlies many long term adverse effects.
Despite the guidelines set by government regulatory authorities, there are cases of drug toxicity, including the recent issues concerning the COX-2 group of pain killers. The toxicity failure rate in the developmental pipeline has remained unimproved over the years contributing to the ever increasing costs of the process. During compound screening, preclinical testing involves both in vitro and animal assays that assess efficacy and potential side effects to predict how the agent will affect humans, but the cost and speed associated with these genotoxicity tests have prohibited the wider use and earlier testing to improve the screening efficiency. For example, a standard first step for detecting mutagenicity is the Ames test, developed almost more than 30 years ago. But even the improved version of the Ames test takes requires 2-4 days to process and costs $4,000 to $5,000 per test to complete. For this reason, Ames tests are often used in later stages of drug development.
Among the required genotoxicity test battery, a large component is evaluation of chromosomal damage, in vitro or in vivo, involving the tk locus using mouse lymphoma L5178Y cells or human lymphoblastoid TK6 cells, the hprt locus using CHO cells, V79 cells, or L5178Y cells, or the gpt locus using AS52 cells. The toxicology field uses the mutation events induced by compounds at these specific loci as representatives of the entire genome, hoping the genetic alterations induced at these loci would be an accurate assessment of the overall DNA damage of the genome, for the simplicity of the model system or just sheer lacking of other efficient and economic ways of evaluation. In an ideal situation, every time a compound's exposure time, dosage, testing cell sampling time or any testing parameter changes, the entire genome, not just a few representative gene loci, of the testing cells or system could be evaluated in detail for damage information with great efficiency and low cost in a standardized format. At least, it would be very beneficial a panel of multi-gene loci, such as one each from every single chromosome or key interested regions, could be analyzed without prohibitive cost and complexity increase. New technology platform that would allow such comprehensive pan-genomic toxicity assessment with efficiency would be greatly desirable.
In the area of DNA damage assessment, decades-old conventional cytogenetic analysis (from karytotyping, G-banding to various forms of FISH) techniques often rely on a spread of metaphase DNA, their resolution is limited to the megabase scale. Interface or fiber-FISH methods attempt to improve the resolution by using relaxed or partially stretched DNA but they are still hard to implement and present challenges when trying to extract quantitative spatial information. Powerful as these techniques are, they suffer from poor control of the processes since they lack consistency and repeatability, hence are ultimately subject to the skill of the technician making them difficult to scale up for faster and cheaper genotoxicity tests.
Other recent attempts trying to improve the linearization of individual DNA molecules using surface “combing”, optical tweezer, fluidic hydrodynamic focusing flow chip formats have reflected the desire to further improve the assay consistency, standardization and cost feasibility. Unfortunately, the methods of the target DNA elongation are not inherently well controlled, the molecule elongation state is often transient, non-uniform and inconsistent, deeming complicated analytical process. Such variability limits the application of this group of single molecule analysis approach for large scale screening of chromosomal DNA structural damages in genotoxicity tests.
Electrophoresis is also employed to separate polymers of various molecular weights based on their varying mobility using gels such as agarose or polyacrylamide. In the case of large polymer fragments, the separation time could take hours or even days. Single cell electrophoresis assays are routinely used to assess the damage of chromosomal DNA induced by toxic agents such as environmental toxins, radiation or agents used in chemotherapy. In a typical assay, termed the comet assay, often used in current genotoxicity tests, the cell is lysed within a gel matrix and then the DNA is electrophoresed and stained with a fluorescent dye. During electrophoresis, DNA fragments migrate away from the cell producing the shape of a comet tail. The geometry of the comet tail is related to the number of double stranded and single stranded breaks within the chromosomal DNA and thus provides a semi-quantitative measure of exposure to toxic agents experienced by the cell. Though this technique offers an assessment of the degree of damage, it is difficult to standardize and the data is subject to interpretation. Also, the fragments of chromosomal DNA remained entangled and cannot be distinguished from each other thus obscuring valuable information regarding the location of breaks or the size of individual fragments.
Other array based approaches such as Comparative Genomic Hybridization (CGH) have progressed in overcoming some aspects of resolution issues in detecting unbalanced genomic structural changes (amplification, deletion not translocation or inversion) however are limited to the issues inherit to comparative hybridization. New-generation sequencing technologies aim to achieve relatively fast sequence data on individual genetic molecules in massive parallel reads; however, molecules analyzed under such techniques must be fragmented into relatively short reads (˜25 bps) with sequence data generated computationally, often by minimizing the tiling path of overlapping reads. A drawback of this approach is that gross genetic changes, usually at much larger scale, can often be obscured because each individual fragment is removed from the context of the entire genome. This is especially relevant in the case of complex genomes with regions of massive repetitive elements and gene copy number polymorphism. Accordingly, such techniques lack the ability to provide information regarding the whole of a genome, as opposed to a discrete region within the genome.
Molecular combing techniques have leveraged work in cytogenetics to generate more detailed analysis at the single molecule level. In molecular combing, DNA is elongated by means of a receding fluid meniscus as a droplet of solution is allowed to dry on a surface. The solute will migrate towards the boundaries of the droplet in a phenomenon known as the ‘coffee-stain’ effect (Deegan 1997). In the case of DNA in a buffer solution, the DNA is tethered to the surface randomly at the boundaries of a liquid phase and then elongated by the shear force of the receding meniscus. Unfortunately, this method of stretching is not inherently well controlled, and DNA samples on different microslides can never be “combed” identically, and there is no way to predict the degree, uniformity of stretching or placement of the molecules on a surface. DNA molecules often overlap each other with imperfect linearization (as they are not physically separated), and their ends often recoil upon themselves once they are released from the meniscus, leaving incompletely-stretched DNA molecules. Such variability accordingly limits the application of the combing approach to large scale screening of patients.
Other attempts to standardize the linearization of individual DNA molecules using fluidic biochip platforms proved relatively inefficient in effecting the desired linearization. DNA would often fold back on itself or even retain its free solution Gaussian coil conformation (essentially, a ball of yarn). The resolution of such techniques, furthermore, is poor, because the elongation of the DNA is often transient, non-uniform and inconsistent, and images used in analysis must be captured while the DNA moves at a high enough velocity to sustain its elongated state. In addition, because these designs are based around a single channel through which flow molecules past an optical detector, their throughput is severely limited.
Accordingly, there is a need for efficient determination of the sizes and composition of fragments of DNA or other macromolecules by linearizing and analyzing such molecules. Such methods would have immediate use in diagnostic and in treatment applications.
It would be desirable to use a minimal amount of sample, perhaps as little as a single cell. This would greatly advance the ability to monitor the cellular state and understand the genesis and progress of diseases such as the malignant stage of a cancer cell or the degree of toxicity of a stimulus leading to apoptosis. There is also a related need for devices capable of performing such methods.